Historically, several approaches have been employed to control the proliferation and infectivity of HIV by identifying various targets that are crucial for the virus to bring about the infection. In the present scenario the following types of anti-viral therapies are commonly known.
a. Anti-Retroviral Therapy Efficiently Controls Viral Proliferation: As a consequence of rapid regeneration rate and high magnitude genetic variation, HIV can rapidly develop drug resistance. To minimize and/or prevent the emergence of drug resistance, anti-retroviral therapy (ART) is typically administered as multi-drug therapy consisting of a minimum of three different drugs often targeting more than one viral factor. The primary objective of ART is to control viral proliferation but not viral eradication. Combination therapy that is administration of three or more drugs is also called highly active antiretroviral therapy (HAART). The viral enzymes reverse transcriptase (RT) and protease are the most common targets for the ART. The anti-RT drugs essentially fall under two classes (1) Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs) and (2) Non-NRTIs (NNRTIs). In the developing countries, HAART consists of combinations of these two anti-RT classes only as they are economically affordable. For more efficient viral control, protease inhibitors (PIs) are also included in the HAART especially in the developed countries. In addition to the above classes, drugs targeting other viral factors or stages are also in use including entry inhibitors, fusion inhibitors and a single integrase inhibitor. Today approximately 25 anti-retroviral inhibitors falling under one of the above classes have been licensed by the Food and Drug Administration for clinical use. Many more new drugs under the above classes or novel classes are at various levels of clinical evaluation (Example: drugs targeting viral maturation or viral factor Tat). In the absence of a promising preventive vaccine, today ART is the only medical intervention strategy for efficient disease management.
b. Anti-Retroviral Therapy Must Target Multiple Viral Factors: To prevent emergence of drug resistance, multiple viral factors must be targeted since the virus cannot generate multiple mutations and yet remain fit. For socio-economic and technical reasons, anti-RT and protease inhibitors constitute the most commonly used HAART regimens. These drugs could also antagonize the various host cellular polymerases and proteases thus manifesting severe side effects often leading to non-compliance which in turn results in drug resistance. Drug resistance to one specific inhibitor could make the virus resistant to all the other members under the same class thus significantly curtailing the options available to the clinician. When drug resistance emerges against the ‘first line therapy, a ‘second line therapy’, consisting of drugs at least from one new class, is usually recommended. Second line therapy inhibitors are, however, expensive and beyond the reach of many subjects especially in developing countries. Switching to second line therapy is also necessitated for drug toxicity reasons.
c. Tat Offers a Good Target for Anti-Viral Inhibitors: Most of the small molecule drugs are likely to possess toxic side effects that differ only in the magnitude of severity. However, drugs that target viral enzymes (polymerases or proteases) are more likely to be toxic since they antagonize host cellular enzymes to variable extent. One possible solution is to target viral factors like Tat that do not have perfect match in the host system, unlike RT and protease. Small molecule inhibitors (SMI) to Tat are less likely to be toxic in comparison to those targeting viral enzymes for specificity reasons. Tat is a viral transactivator that controls gene expression regulation from the viral promoter, the long-terminal repeat (LTR). Although the LTR is functional in the absence of Tat, especially soon after viral infection where Tat is yet to be made, under the influence of Tat, the LTR is one of the strongest mammalian promoters known often up-regulating gene expression 100-1000 fold. Tat also constitutes an important molecular switch between active viral proliferation and viral latency. Absence of Tat in the cell pushes the virus into a genetically silent mode called viral latency. A latent viral infection is recalcitrant to retroviral therapy and immune response thus posing a serious challenge to viral eradication efforts. Given that no host equivalent of Tat exists in the cell and that Tat plays an important role in viral gene expression and establishment and maintenance of viral latency, developing SMI to Tat is extremely important. Additionally, inhibiting Tat broadens the range of ART by adding a novel viral target thus minimizing the emergence of drug resistance.
d. Small Molecule Inhibitors to Tat are not Available: Of the various kinds of Tat inhibitors, including siRNA, intrabodies, aptamers etc, only small molecule inhibitors (SMI) have a potential of practical application to the clinic. SMI have the following advantages (1) unlike other inhibitors, chemical libraries consisting of a very wide range of molecules are available for SMI, (2) furthermore, SMI have a superior reach in that small molecules can reach each and every infected cell in the body, (3) additionally, SMI have an advantage of economically low cost but large scale synthesis. Despite all the merits, paradoxically, no anti-Tat SMI are available today. Worse, there are no drugs at any level of clinical trial targeting Tat. In the 1990s a few pharmaceutical companies identified a few molecules with anti-Tat properties but for unknown reasons, these molecules did not progress to clinical trials. Ro 24-7429, the Hoffmann-La Roche is one such example. There have been several reports on anti-Tat inhibitors in the medical literature; however, none of them reached an advanced stage of pharmaceutical development. Despite thousands of scientific publications on the HIV Tat, the knowledge is not translated into practical drug development.
e. Limitation of the Existing HTS (High Throughput Screening) Assays: Tat is a viral transactivator that controls gene expression from its own promoter, the viral LTR. In the presence of Tat, the LTR makes 100 to 1000 times more viral protein for instance. Tat also controls establishment and maintenance of viral latency. The property of gene expression control of the viral LTR is exploited to develop the standard HTS assay for Tat regardless the nature of the inhibitor. Typically, a reporter gene like green fluorescent protein (GFP) is placed under the control of HIV-1 LTR on a DNA plasmid. Mammalian cells with stably integrated LTR-GFP plasmid could be established and such are called reporter cell lines. Reporter cell lines express GFP in the presence of Tat (FIG. 1) and if Tat is blocked expression of GFP is also inhibited. A Tat-inducible GFP expressing cell line could used in a high throughput screening (HTS) to screen for molecules that may possess anti-Tat or anti-HIV properties.
The most serious limitation of the standard reporter cell lines is that they cannot discriminate between cytotoxic molecules and anti-viral compounds since in both of these events GFP is likely to be down regulated (FIG. 2). For instance, an SMI that interferes with the regular process of cellular protein synthesis could results in the down regulation of GFP expression but not due to anti-viral property. Such a molecule also will be cytotoxic resulting in cell death or tissue injury. Given the complexity of the cellular metabolism, any SMI that interferes with any of the essential biochemical pathways is likely to be cytotoxic and often may lead to GFP down-regulation offering false hits. Such molecules will be proved unsuccessful after enormous effort of characterizing them. This is one reason why most of the drug screen assays failed to identify SMI that are non-cytotoxic and anti-viral for HIV and Tat.
In the absence of a preventive vaccine, chemotherapy is the only available option today for effective disease management for HIV/AIDS. HIV has a potential for generating extraordinarily great levels of genetic variation that leads to rapid drug resistance. Furthermore, the most commonly used anti-viral drugs are highly toxic given that these drugs primarily target viral polymerase and protease, the enzymes that have host equivalents. Additionally, development of drug resistance to one specific drug could lead to broad-level resistance to the entire class thus making any other drug under the same class ineffective. It is therefore critical to identify drugs to counter viral drug resistance. Importantly, the drugs must target less commonly employed viral targets to widen the effectiveness of the anti-retroviral therapy. To this end, targeting viral factors is less likely to be toxic given the absence of host homologues of these viral factors. Despite these merits, essentially there have been no anti-viral factor drugs approved by FDA today in the market, except reltegravir. The traditional method of structure-based-drug-design has not been applicable to some viral factors given that the crystal structure of the factors could not be determined owing to its structural flexibility. Several of these drugs are at various levels of evaluation in clinical trials and none yet reached the clinic. The present disclosure presents aspects which overcome the demerits observed in the prior research in this field of technology.